Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.
The lymphocytes within the vertebrate immune system recognize and respond to an enormous number of different antigens. Antigen recognition for B and T lymphocytes, respectively, is effected through the variable domains of immunoglobulin (Ig) or T cell receptor (TCR) molecules. Genes that encode these variable regions are assembled during the early stages of B and T lymphocyte differentiation from germline variable (V), diversity (D) and joining (J) gene segments by a process referred to as VDJ recombination. The enzymatic activity responsible for assembling these gene segments has been assayed within permanent precursor lymphoid cell lines through the use of recombination substrates that contained unrearranged V, D, or J segments plus selectable marker genes (reviewed by Blackwell and Alt, 1989). These types of assay demonstrated that the conserved recognition sequences (RS) that flank all antigen receptor variable region gene segments are sufficient to target VDJ recombinase assembles all antigen receptor gene segments (Yancopoulos, et al., 1986). In addition, assays of cell lines demonstrated that VDJ recombinase activity is expressed specifically in precursor (pre) B and T cells and not in non-lymphoid cells or in cells that represent more mature stages of the lymphocyte lineages (Blackwell, et al., 1986; Lieber, et al., 1987; Schatz and Baltimore, 1988).
Two genes that synergistically confer fibroblasts with ability to specifically rearrange transfected VDJ recombination substrates have been isolated and referred to as the recombination activating genes (RAG) 1 and 2 (Shatz, et al., 1989; Oettinger, et al., 1990). These two genes are evolutionarily conserved in vertebrates and closely linked in the chromosomes of mice, humans, and chickens (Shatz, et al., 1989; Oettinger, et al., 1990). The precise function of the RAG gene products has not been unequivocally elucidated, however, it is generally believed that they may encode the tissue specific components of the VDJ recombination system (Chun, et al., 1991). In the latter context, high level expression of the RAG-1 and RAG-2 genes has been found only in primary lymphocyte differentiation organs (eg. the thymus) and in cell lines that represent precursor stages of B and T cell development (Shatz, et al., 1989; Oettinger, et al., 1990). However, low level expression of RAG-1 and RAG-2 has been observed in a number of different organs (Chun, et al., 1991; Carlson, et al., 1991) fueling speculation that some type of site-specific recombination process may occur in other developmental processes.
The VDJ recombination process is believed to involve multiple activities, including recognition of the RS, endonucleolytic activity that site-specifically cleaves at the border of RS and adjacent gene segments, potential exonucleolytic and nucleotide-addition activities, polymerase activities, and ligase activity to join the free ends (reviewed by Blackwell and Alt, 1989 and Lewis and Gellert, 1989). Several of these activities, including RS recognition and cutting, are likely to involve lymphocyte specific activities, while others may reflect more generally expressed activities recruited by the lymphocyte specific components. A potential example of the latter is the activity encoded by the gene affected by the murine scid mutation (reviewed by Bosma and Carroll, 1991). Although mice homozygous for this mutation are impaired in the final, joining step of the VDJ recombination process (Malynn, et al., 1988; Lieber, et al., 1988; Blackwell, et al., 1989) resulting in the severe combined immune deficient (SCID) phenotype, the activity affected by the scid mutation appears to also be more generally involved in the DNA repair process (Fulop and Philips, 1990; Biedermann, et al., 1991., Hendrickson, et al., 1991).
Previous studies and applications using the SCID mice have been based on mice homozygous for the scid mutation (for general review, see McCune et al., 1989; Bosma and Carroll, 1991). Such scid mutated mice are severely deficient in functional B and T lymphocytes. The mutation appears to impair the recombination of antigen receptor genes and thereby causes an arrest in the early development of B and T lineage-committed cells; other hematopoietic cell types appear to develop and function normally.
One of the problems of scid mutated mice is that the arrest in lymphocyte development is not absolute; some young adult SCID mice are leaky and generate a few clones of functional B and T cells. By 10-14 months of age, virtually all SCID mice are leaky.
The recombination activating genes I and 2 (RAG-1 and 2) synergistically confer VDJ recombination activity to non-lymphoid cells. To assess RAG-2 gene function in normal physiology, we have deleted a large portion of the RAG-2 coding region in an embryonic stem cell line and used these cells to generate mice that harbor the mutation in their germline. Homozygous mutants are viable but fail to produce mature B or T lymphocytes even at several months of age. Very immature lymphoid cells were present in primary lymphoid organs of the homozygous mutants as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, we could not detect rearrangements of immunoglobulin (Ig) or T cell receptor loci in cells of primary lymphoid organs or in homozygous mutant A-MuLV transformed pre-B cell lines. Lack of VDJ recombination activity in the mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate VDJ rearrangement leading to a novel SCID phenotype. Furthermore, the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, indicating the VDJ recombinase activity, per se, is not required for development of cells other than lymphocytes.
SCID mice are susceptible to various infectious agents due to the absence of an effective immune system. SCID mice are highly susceptible to Pneumocystis carinii, an parasitic micro-organism which causes severe opportunistic infections in immune deficient humans, including HIV-1 infected humans (Roths et al., 1990). The SCID model should facilitate analysis of the factors determining P. carinii resistance, and it may provide a disease model in which therapeutic regimens can be tested.
Studies have shown that SCID mice are also susceptible to infections of spirochete Borrelia burgdorferi, which causes lyme borreliosis (Schaible et al., 1989). Such infections cause lyme arthritis and carditis. SCID is not only useful in propagating infective B. burgdorferi, it would be a good model for elucidating the role of cellular and humoral immune responses in the pathogenesis of lyme borreliosis. Different drugs or therapeutic protocols may be tested in this animal system.
Tumors can grow in SCID mice (summarized in Philips et al., 1989). Tumor tissues including retinoblastoma and osteogenic tumors, acute lymphoblastic leukemia, urologic malignant tumors, and human melanoma can grow in SCID mouse by engraftment. Depending on the types, tumors may be introduced intravenously, intraperitoneally or subcutaneously.
Human lung tumor growth has been established in the lung and subcutaneous tissue of SCID mice. The growth of a human lung tumor cell line may serve as a metastatic model in which to investigate patient lymphocyte tumor infiltration, and therapeutic and diagnostic efficacies of antitumor antibodies.
Human yolk sac tumor (YST-2) grew rapidly to enormous sizes in all SCID mice after both subcutaneous and intraperitoneal transplantation, while only half of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (Nomura, et al., 1990). Furthermore, transplanted tumors metastasized spontaneously to distant organs such as the lung, liver, kidney, pancreas, and spleen, in scid mutated mice, while metastases were not found in athymic nude mice. Similar results were observed in scid mutated mice and scid-nude double mutant mice with human classic seminoma which has been neither transplantable nor metastatic in athymic nude mice. Thus, SCID mice provide an invaluable experimental system for investigating the mechanism of metastasis, which is the most important and life-threatening problem in cancer patients.
Human germinal tumors were ectopically transplanted to SCID mice, and metastasis mimicking what were found in human were observed. It was also found that ectopically transplanted tumors spontaneously metastasized to distant organs in SCID mice but less frequently in leaky SCID mice, while metastasis has never been found in nude mice (Nomura T., et al., 1991).
SCID mice also provides an efficient and reproducible model to study the pathogenesis of children acute lymphoblastic leukemias and provides a suitable system for evaluating therapy. Upon intraperitoneal transfer, T cells from acute lymphoblastic leukemias spread hematogenously and infiltrate the non-lymphoid and/or lymphoid organs with a pattern reminiscent of the human clinical disease (Cesano et al., 1991).
The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. Human cutaneous T-cell lymphoma has been established in SCID mice. In addition, primary human acute leukemia has been grown in SCID mice (DeLord et al., 1991).
Overall, the SCID mouse provides a useful in vivo model of human tumor establishment, progression, metastasis, and treatment.
SCID mice with defects in the maturation of T and B cells have provided a novel experimental system in which to study normal lymphoid differentiation and function in mouse and man (reviewed in McCune, J. M., 1991). A useful SCID-hu mouse is established by implanting human cells or tissues into the SCID mouse (reviewed in Mosier, D. E., 1990).
Via engraftment with human lymphoid progenitors, the SCID-hu mouse has been used to study infections of human lymphoid cells with the human immunodeficiency virus, HIV-1. The mouse may either first receive the engraftment of human lymphoid progenitors cells and subsequently be inoculated with the HIV virus or it may be engrafted directly with the virus-infected progenitor cells. The mouse may enable determination of how progressive infection occurs in defined CD4 lymphoid and myelomonocytic cell populations and may also be used to analyze the efficacy of antiviral drugs and vaccines, including the drug AZT. It has been shown that the animals were protected in dose ranges similar to those used in man. This animal model may now be used as an efficient intermediate step between the laboratory and the clinic to study the infectious process in vivo and to best select efficacious antiviral compounds against HIV (Kaneshima, et al., 1991).
Similarly, Epstein Bar Virus-related lymphoproliferative disorders can also be modeled by the transfer of adult peripheral blood mononuclear cell to SCID mice. Other viruses which can infect lymphocytes can be similarly studied. Once such a system is established, various kinds of drug and different schemes of treatment can be tested.
The SCID-hu mice have also been used to evaluate cytokine-induced killer cells with potent antitumor cell activity (Schmidt-Wolf, et al., 1991).
Fetal liver cells have been transplanted in SCID mice. Coimplantation of small fragments of human and fetal liver into immunodeficient SCID mice resulted in the formation of a unique structure (Thy/Liv). Thereafter, the SCID-hu mice showed reproducible and long-term reconstitution of human hematopoietic activity. For periods lasting 5-11 months after transplantation, active T lymphopoiesis was observed inside the grafts and cells that were negative for T cell markers were found to have colony-forming units for granulocyte/macrophage and erythroid burst-forming unit (BFU-E) activity.
The SCID-hu mouse can also be used in the analysis of the growth factors which regulate hematopoiesis (McCunne, et al., 1989). When fetal liver cells are administrated to the SCID-hu mouse intravenously in the absence of microenvironmental stromal cells and the growth factors they produce. The production of mature cells is time-limited. If human stem cells can be isolated, they will in turn serve as assays, either in vivo or in vitro, for the identification of those factors which regulate self-renewal and/or differentiation. This line of investigation has in the past been hampered for lack of a suitable assay. The SCID-hu mouse represents not only a means by which to purify stem cells and their progeny but also an assay system in which to monitor growth and differentiation.
It has been shown that bone marrow can be transplanted into SCID mice (Dorshkind, et al., 1989; Dorshkind, et al., 1986). The SCID mice have also been used as a model to identify and quantify myeloid and lymphoid stem cells (Fulop, G. M. 1989). SCID mice were also used to study the function of natural killer (NK) cells in bone marrow transplant (Murphy, W. J., 1989). SCID mouse contain normal NK cells and their progenitors and, therefore, provide a lymphoid-free system in which to study NK effector functions.
The feasibility of reconstitution of SCID mice with lymphocytes from normal mice and the possible engraftments with peripheral blood lymphocytes from other species render SCID mice ideal for assessing the importance of lymphocytes in control of pathogens, immune surveillance and reproduction.
Moreover, human and rat islet tissues have been transplanted to SCID-hu mice, and success has been observed (London, et a1.,1991). Therefore, SCID-hu mice are useful in studying the effects of human immune response mediated by T and B lymphocyte against the islet tissue.
Though most of the above applications were done in scid mutated mice, it is an important objective of this invention that the RAG-2 deficient animal have an improved SCID phenotype and, therefore, the same application.
In scid mutated mice, leakiness may limit their applications. Scid mutated mice were first recognized as leaky on the basis of serum Ig in 2-25% of young adult mice (Bosma, et al., 1988). Though the molecular basis for the leaky SCID phenomenon is not yet clear, recent evidence suggests that leaky lymphocyte clones may reflect rare genetic events that enable a given SCID lymphoid progenitor and progeny to form a normal VDJ coding joint at normal frequency. A few clones of antigen receptor positive B and T lymphocytes do appear in a variable proportion of young adult SCID mice and in virtually all old SCID mice (Carroll et al., 1989; Carroll and Bosma 1988). As stated previously, some tumor implantations are not as successful because of this leakiness.
The recombinant RAG-2 deficient mice disclosed in this invention do not have any leaky phenotype and therefore are more advantageous than the scid mutated mice.
Another advantage of this invention is that RAG-2 mutation affects an earlier stage of VDJ recombination, and therefore, RAG-2 may be applied to areas which cannot be accommodated by scid mutated mice.
A third advantage may be that RAG-2 deficient mice appear to affect only lymphoid cells. The scid mutation has deleterious effects on general DNA repair mechanism and thus affect many different cell lineage.
Finally, scid mutated mice are generally difficult to propagate. To this point, RAG-2 deficient mice are able to propagate readily.